Novel pharmaceutical formulations comprising aminoalkyl phosphorothioates

ABSTRACT

The present invention provides novel pharmaceutical compositions comprising aminoalkyl phosphorothioate compounds in combination with surfactants, hydrotropes and chelating agents. The compositions are well-suited for subcutaneous administration.

1. FIELD OF THE INVENTION

[0001] The present invention relates to novel pharmaceuticalformulations comprising aminoalkyl phosphorothioate compounds. Theformulations preferably include surfactants, hydrotropes and chelatingagents to, inter alia, enhance the chemical and biological properties ofthe aminoalkyl phosphorothioate compounds.

2. BACKGROUND OF THE INVENTION

[0002] The compound S-2-(3-aminopropylamino)ethyl dihydrogenphosphorothioate (which is also known as amifostine, ethiofos, Ethyol®,NSC 296961, and WR-2721 and which will hereinafter be referred to as“amifostine”) and other aminothiol compounds are disclosed in U.S. Pat.No. 3,892,824. These compounds were originally developed asantiradiation agents (radio-protectants), in particular to be used priorto exposure to x-ray or nuclear radiation, to protect against theharmful effects of such exposure which may be encountered duringmilitary conflicts.

[0003] In addition to its utility as a military antiradiation agent,amifostine has demonstrated excellent utility as a non-militaryradioprotectant and chemoprotectant, i.e., as a protectant administeredprior to therapy to reduce the undesirable adverse effects which ariseduring the use of chemotherapy and radiation therapy in the treatment ofcancer. Nygaard et al., eds, Radioprotectors and Anticarcinogens,Academic Press, Inc., New York, pp. 73-85 (1983); Grdina et al.,Carcinogenesis (London) 6:929-931 (1985). In addition, these compoundshave been reported to afford protection against the adverse effects ofchemotherapeutic agents, for example, alkylating agents such ascisplatin, when administered before or concurrently with thechemotherapeutic agent. Jordan et al., Exp. Mol. Pathol. 36:297 (1982);Doz et al., Cancer Chemother. Pharmacol. 28:308 (1991). Similarly, ithas been reported that amifostine has been used experimentally prior totherapy to protect HIV-infected patients (AIDS) from the harmful sideeffects of 3′-azido-3′-deoxythymidine (AZT) therapy. InternationalPublished Application WO 90/14007, published Nov. 29, 1990. Amifostineand its derivatives have been shown to exert these reported protectiveeffects without affecting the beneficial properties of the administeredtherapeutic agents. This is, in the case of chemotherapy, believed to bedue to the selective uptake of the protective thiol and othermetabolites into normal tissue. Yuhas, Cancer Res. 40:1519-1524 (1980);Yuhas, Cancer Treat. Rep. 63:971-976 (1979).

[0004] Amifostine is indicated to reduce the cumulative renal toxicityassociated with repeated administration of cisplatin in patients withadvanced ovarian or non-small cell lung cancer. Physicians' DeskReference 53^(rd) ed., p. 513-515 (1999).

[0005] Pharmaceutical compositions containing amifostine and a chelatingagent to enhance gastrointestinal tract absorption of the activecompound have been reported. Such compositions, suitable for oral orrectal administration, are described in U.S. Pat. No. 5,167,947.Amifostine is also being developed for subcutaneous administration. See,International Publication No. WO98/34622. In its most common use,however, amifostine is administered parenterally, including by bolusinjection and intravenous infusion. Since parenteral routes circumventthe protective barriers of the human body, exceptional purity of thedosage form must be achieved. Because the dosage form must be free ofmicroorganisms and insoluble particulates, the process used in preparingit must embody Good Manufacturing Practices (“GMP”) that will produceand maintain the required quality of the product in terms of sterilityand therapeutic effectiveness. Sterility is especially important in thetreatment of cancer and AIDS patients, because in many instances theyare already immuno-compromised and therefor highly susceptible toinfections.

[0006] Amifostine has been sold as a sterile amorphous lyophilizedpowder containing active ingredient and mannitol. The mixture wassupplied as a single use 10 ml vial containing 500 mg of amifostine and500 mg of mannitol, and required reconstitution for intravenousinfusion. Physicians'Desk Reference, 51^(st) ed. (1997) p. 485-486.Presently, a sterile crystalline dosage form of amifostine is sold underthe trade name Ethyol®. Physicians'Desk Reference, 53^(rd) ed. (1999) p.513-515. A crystal structure and preparation of a dosage form, whichexhibits greater thermal stability than the previously sold amorphousdosage form, is described by U.S. Pat. Nos. 5,424,471 and 5,591,731.

[0007] Intravenous administration of amifostine suffers from severalserious drawbacks. First, administering compounds intravenously isextremely inconvenient, particularly when a daily dosing schedule forseveral weeks, or potentially several months in the case ofmyelodysplastic syndrome (“MDS”), is necessary, requiring a skilledpractitioner to administer the dose. Second, when administeredintravenously, patients suffer from dose-dependent undesirableside-effects such as nausea, vomiting, emesis and hypotension, as wellas flushing or feeling of warmth, chills or feeling of coldness,dizziness, somnolence, hiccups and sneezing. A decrease in serum calciumconcentration is a known pharmacological effect of intravenouslyadministered amifostine. Allergic reactions ranging from mild skinrashes to rigors have also rarely occurred in conjunction withintravenously administered amifostine. At present, there are no knownmethods, other than co-administering agents such as anti-emetics, ofreducing or avoiding these undesirable side effects. Third, there arerelated costs associated with intravenous administration, includingpersonnel, equipment and medical measures to attenuate side effects.

[0008] Therefore, there remains a need for pharmaceutical deliverysystems which are capable of delivering amifostine and related compoundsto the patient in an effective, efficient and economical manner.

3. SUMMARY OF THE INVENTION

[0009] The present invention provides novel pharmaceutical formulationscomprising aminoalkyl phosphorothioate compounds. The pharmaceuticalcompositions of the present invention are efficacious formulationswherein the biological and/or chemical properties of the active agentare enhanced by the addition of ingredients including, but not limitedto, one or more of the following: surfactants, chelating agents andhydrotropes. These additional formulation ingredients are considered toserve, individually or in combination, inter alia, to increase thebiological activity or chemical properties of the active compound.

[0010] The novel pharmaceutical formulations of the invention may beadministered to a mammal, including humans, to treat or prevent avariety of disorders associated with radiation or chemotherapy, and in amanner which decreases or reduces undesirable side effects associatedwith the compounds. In preferred embodiments, the formulations of theinvention comprise amifostine, and the formulations are administeredsubcutaneously.

[0011] One aspect of the invention relates to pharmaceuticalformulations comprising an aminoalkyl phosphorothioate and a surfactantalone. Such formulations, in accordance with the present invention, areconsidered to have improved chemical and biological properties. Theinvention also encompasses pharmaceutical formulations comprising anaminoalkyl phosphorothioate, a surfactant and a hydrotrope, wherein thehydrotrope, inter alia, enhances the solubility of the formulationcomposition. The invention further encompasses pharmaceuticalformulations comprising an aminoalkyl phosphorothioate, a hydrotrope anda chelating agent. The formulations of the invention having a chelatingagent are considered to have enhanced biological properties. Thisformulation is also considered to exhibit beneficial chemical andmanufacturing properties, including, inter alia, improving freeze-dryingprocedures and freeze-drying itself. The invention further encompassespharmaceutical formulations comprising an aminoalkyl phosphorothioate, asurfactant, a hydrotrope, and a chelating agent.

[0012] The subcutaneous route of administration leads to a limitation inthe volume of the pharmaceutical formulation used due to distention ofthe skin at the injection site and associated pain. Thus, the presentinvention has a further benefit of increasing the solubility of theactive compounds in the pharmaceutical compositions of the invention,such that a greater amount of active compound can be administered perdose, without additional pain at the injection site.

[0013] Another aspect of the invention relates to sterile dosage formsof the above-described pharmaceutical compositions. The pharmaceuticalcompositions of the present invention may be in liquid or solid formssuitable for reconstitution such as freeze-dried or lyophilized forms,although the invention includes non-freeze-dried solids. Thepharmaceutical composition may comprise a single dosage form whichcomprises all the components of the composition. Alternatively, theinvention encompasses a dosage form kit wherein the aminoalkylphosphorothioate compound and the remaining components of theformulation composition are provided in separate containers, and asolution for use is prepared by combining the formulation ingredientswith a suitable carrier, such as sterile water prior to administration.

[0014] The pharmaceutical compositions of the invention comprisingaminoalkyl phosphorothioate compounds may be adapted for administrationto a patient in a number of ways, including, but not limited toparenteral (including subcutaneous, intravenous and intramuscular); ororal; mucosal (including buccal, sublingual, vaginal and rectal);topical, transdermal and the like. Subcutaneous administration ispreferred.

[0015] The present invention is further directed to novel pharmaceuticalcompositions comprising aminoalkyl phosphorothioate and other activeingredients. Finally, the pharmaceutical compositions of the inventionmay also include solvents and co-solvents, excipients, antioxidants,carriers, diluents, buffers, bulking agents, solubilizers, wettingagents, suspending agents, emulsifiers and thickening agents.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1A is a graph comparing the weight in mice, followingadministration of saline solution and subsequent irradiation; 50 mg/mlamifostine solution and subsequent irradiation; and saline solutionwithout irradiation (control). FIG. 1B compares the total mucositisscores.

[0017]FIG. 2A is a graph of the weight in mice following administrationof 50 mg/ml amifostine and 0.075% sodium deoxycholate, followed byirradiation, to 5 subjects, 0 to 25 days following irradiation. FIG. 2Bdepicts the total mucositis score of this formulation.

[0018]FIG. 3A is a graph of the weight in mice following administrationof 50 mg/ml amifostine, 2% EDTA and 2% sorbitol, followed byirradiation, to 5 subjects, 0 to 25 days following irradiation. FIG. 3Bdepicts the total mucositis score of this formulation.

[0019]FIG. 4A is a graph comparing the weight in mice followingadministration of a formulation comprising 50 mg/ml amifostine and 0.2%EDTA followed by irradiation; and a formulation comprising 50 mg/mlamifostine and adjusted to pH=7 followed by irradiation. FIG. 4Bcompares the total mucositis scores of the formulations.

[0020]FIG. 5A is a graph comparing the weight in mice followingadministration of: a 50 mg/ml amifostine solution adjusted to pH=7followed by irradiation; a formulation comprising 50 mg/ml amifostineand 0.075% sodium deoxycholate (NaDC), followed by irradiation; and aformulation comprising 50 mg/ml amifostine, 2% EDTA and 2% sorbitol(EDSO), followed by irradiation. FIG. 5B compares the total mucositisscores of the formulations.

5. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides novel pharmaceutical compositionscomprising aminoalkyl phosphorothioate compounds. The pharmaceuticalcompositions may comprise a surfactant and/or a hydrotrope and/or achelating agent to yield a aminoalkyl phosphorothioate containingcomposition having enhanced chemical and biological properties. Theinvention is based, in part, on the surprising and unexpected discoverythat the chemical, physical and/or biological properties ofpharmaceutical compositions comprising aminoalkyl phosphorothioatecompounds are enhanced by the addition of a surfactant and/or ahydrotrope and/or a chelating agent and/or an enzyme (such ashyaluronidase). Such enhancement can improve or facilitate themanufacture and/or use of the compositions.

[0022] While not intending to be limited by any theory, it is believedthat increased biological activity may be brought about by the enhancedsolubility and rate of dissolution of the active compounds in thepharmaceutical compositions. Enhanced solubility is especially desirablewhen the pharmaceutical composition is to be administeredsubcutaneously, due to the limitation on the volume of the compositionwhich may be used. The pharmaceutical compositions of the invention maydemonstrate additional beneficial properties by increasing theabsorption of vitamins and other nutrients.

[0023] The formulations of the invention can be used to deliver to amammal, including a human, the active compounds described herein.Further, the compositions are suitable for treatment or protection ofpatients suffering from any disorder for which the aminoalkylphosphorothioate compounds are known or proposed to be useful. Theformulations may be delivered to a patient using numerous routes ofadministration including, but are not limited to, inhalation,transdermal, oral, rectal, transmucosal, intestinal and parenteraladministration, including intramuscular, subcutaneous, intravenousinfusion or bolus injection. The referred route of administration usingthe compositions of the invention is via subcutaneous administration.The formulations of the invention comprising an aminoalkylphosphorothioate and, optionally, a surfactant and/or a hydrotropeand/or a chelating agent provide advantageous methods of treatment orprotection (e.g., prevention of disease) of patients by demonstratingimproved biological properties compared to formulations comprising onlythe active aminoalkyl phosphorothioate. These formulations are thusbelieved to address the need for a more efficacious dosage form foraminoalkyl phosphorothioate compounds.

[0024] The invention further provides sterile dosage forms wherein theformulation is provided in a lyophilized form, suitable forreconstitution and administration to a patient. The invention may alsobe provided in a form wherein the active compounds and the otheringredients of the formulation are each provided in solidnon-lyophilized or lyophilized form, separate from each other. Theseingredients are then reconstituted and/or solubilized in a suitablesterile liquid and combined to produce the pharmaceutical formulation,which is then suitable for administration to a patient.

[0025] The pharmaceutical compositions of the present invention compriseaminoalkyl phosphorothioate compounds in combination with a surfactantand/or a hydrotrope and/or a chelating agent, and, optionally, otheringredients. Suitable pharmaceutical ingredients are described in moredetail below.

[0026] 5.1 Aminoalkyl Phosphorothioates and Related Compounds

[0027] The active compounds that can be used within the formulations ofthe present invention include, but are not limited to, amifostine(WR-2721), as well as salts, hydrates, active metabolites, pro-drugs,and functional derivatives or analogues thereof. More specifically, theactive compounds of the formulations of the invention include, but arenot limited to, all pro-drugs and metabolites of amifostine andpro-drugs of the active metabolites. Thus, compounds known to theskilled artisan to be suitable for administration to humans and known tobe metabolites or otherwise converted into active thiols includingmetabolites such as WR-1065 and WR-33278 (disulfide) and the orallybioavailable WR-151327 and its active thiols, including metabolites suchas WR-151326 and its corresponding disulfide, are encompassed within theformulations.

[0028] Similarly, described herein are aminothiols that exhibit activitysimilar to that of amifostine or its metabolites. Preferably, thesecompounds are structurally related to amifostine. Alternatively, theactive compounds are pro-drugs that are metabolized in vivo to abiologically active agent. Specific examples are illustrated herein.

[0029] Aminothiol compounds which can be used in the formulations of thepresent invention are represented by the following formula (I):

R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂  (I)

[0030] wherein R₁ is hydrogen, C₅-C₇ aryl, C₂-C₇ acyl, or C₁-C₇ alkyl;R₂ is hydrogen, PO₃H₂ or R₃, wherein R₃ is R₁NH(CH₂)_(n)NH(CH₂)_(m)S—; nand m are each an integer from 1 to 10; and preferably an integer from 2to 6.

[0031] The formulations of the present invention also encompass the useof pharmaceutically acceptable salts and hydrates of the compounds offormula (I) above.

[0032] Preferred compounds useful in the formulations of the inventionare the S-ω(ω-amino-alkylamino)alkyl dihydrogen phosphorothioateanalogues represented by the formula:

R—NH—(C_(n)H_(2n))—NH—(C_(m)H_(2m))—S—PO₃H₂

[0033] wherein R is hydrogen or an alkyl group comprising 1 to 7 carbonatoms and m and n independently have a value of from 1 to 10, preferably2 to 6.

[0034] The chemical structure of amifostine (WR-2721) can be depicted asfollows:

H₂N—(CH₂)₃—NH—(CH₂)₂—S—PO₃H₂.

[0035] One preferred metabolite of amifostine is a dephosphorylated freethiol form known as WR-1065 (chemical nomenclature:S-2-(3-aminopropylamino) ethanethiol), which can be depicted as follows:

H₂N—(CH₂)₃—NH(CH₂)₂—SH.

[0036] Another preferred metabolite of amifostine is its disulfide,known as WR-33278 (chemical nomenclature:[2-[(aminopropyl)amino]ethanthiol]-N,N′-dithioidi-2,1-ethanediyl)bis-1,3-propanediamine),which can be depicted as follows:

H₂N—(CH₂)₃—NH—(CH₂)₂—S—S—(CH₂)₂—NH—(CH₂)₃—NH₂.

[0037] A preferred analogue of amifostine is the compound designated asWR-151327 (chemical nomenclature:1-propanethiol-3-[[3-(methylamino)propyl]amino]-dihydrogenphosphorothioate), which can be depicted as follows:

CH₃NH(CH₂)₃NH(CH₂)₃SPO₃H₂.

[0038] Another preferred analogue of amifostine is the compounddesignated WR-151326, a dephosphorylated free thiol form of WR-151327having the chemical structure: CH₃NH(CH₂)₃NH(CH₂)₃SH.

[0039] Other specific compounds suitable for use in the formulations ofthe present invention include, but are not limited to:

[0040] S-1-(aminoethyl)phosphorothioic acid (WR-638);

[0041] S-[2-(3-methylaminopropyl)aminoethyl]phosphorothioate acid(WR-3689);

[0042] S-2-(4-aminobutylamino)ethyl phosphorothioic acid (WR-2822);

[0043] 3-[(2-mercaptoethyl)amino]propionamide p-toluene-sulfonate(WR-2529);

[0044] S-1-(2-hydroxy-3-amino)propyl phosphorothioic acid (WR-77913);

[0045] 2-[3-(methylamino)propylamino]ethanethiol (WR-255591);

[0046] S-2-(5-aminopentylamino)ethyl phosphorothioic acid (WR-2823);

[0047] 1-[3-(3-aminopropyl)thiazolidin-2-Y1]-D-gluco-1,2,3,4,5pentane-pentol dihydrochloride (WR-255709).

[0048] Additional aminothiols suitable for use in the formulations ofthe present invention include, but are not limited to,S-2-(3-ethylaminopropylamino)ethyl dihydrogen phosphorothioate,S-2-(3-aminopropylamino)-2-methylpropyl dihydrogen phosphorothioate,S-2-(2-aminoethylamino)-2-ethyl dihydrogen phosphorothioate,S-2-(4-aminobutylamino)-2-ethyl dihydrogen phosphorothioate,S-2-(5-aminopentylamino)-2-ethyl dihydrogen phosphorothioate,S-2-(6-aminohexylamino)-2-ethyl dihydrogen phosphorothioate,S-2-(2-methylaminoethylamino)-2-ethyl dihydrogen phosphorothioate,S-2-(3-methylaminopropylamino)-2-ethyl dihydrogen phosphorothioate, andS-3-(3-methylamino-propylamino)-3-propyl dihydrogen phosphorothioate(WR-151327) and pharmaceutically acceptable salts thereof. Preferably,the aminothiol is amifostine, WR-1065, WR-33278, WR-151327 or WR-151326;most preferably it is amifostine.

[0049] Amifostine, and many of its salts, analogues and derivativesthereof suitable for use in the formulations of the invention arecommercially available, or can readily be prepared using standardtechniques. The aminothiol compounds useful in the formulations of theinvention may be prepared by methods known in the art (see, e.g.,Cortese, 1943, Organic Synthesis pp. 91-93, Coll. Vol. II, Blatt, Ed.,John Wiley & Sons, Inc., New York, N.Y.; Akerfeldt, 1960, Acta Chem.Scand. 14:1980; Piper et al., 1966, Chem. Ind. (London):2010). Certainaminothiol compounds, as well as methods of synthesizing such compounds,are described in detail in U.S. Pat. No. 3,892,824 to Piper et al., U.S.Pat. Nos. 5,424,472 and 5,591,731, both to Kennedy et al., and WO96/25045, each of which is incorporated herein by reference in itsentirety.

[0050] The aminothiol compounds useful in the formulations of theinvention may be in the form of free acids, free bases, orpharmaceutically acceptable addition salts thereof. Such salts can bereadily prepared by treating an aminothiol compound with an appropriateacid and/or base. Such acids include, by way of example and notlimitation, inorganic acids such as hydrohalic acids (hydrochloric,hydrobromic, hydrofluoric, etc.), sulfuric acid, nitric acid, phosphoricacid, etc. and organic acids such as acetic acid, propanoic acid,2-hydroxyacetic acid, 2-hydroxypropanoic acid, 2-oxopropanoic acid,propandioic acid, butandioic acid, etc. Conversely, the salt can beconverted into the free base form by treatment with alkali.

[0051] The aminothiol compounds useful in the formulations of theinvention, as well as the pharmaceutically acceptable addition saltsthereof, may be in a hydrated, solvated or anhydrous form. Methods ofpreparing such forms will be apparent to those skilled in the art oforganic chemistry.

[0052] For any mode of administration, the actual amount of activeaminoalkyl phosphorothioate compound to be delivered, as well as thedosing schedule necessary will depend, in part, on such factors as thebioavailability of the active compound to be used (and/or an activemetabolite thereof), the patient's condition, age and height, thedisorder being treated, the desired therapeutic effect (e.g.,prevention), the frequency and route of administration, and otherfactors that will be apparent to those of skill in the art. The actualamount delivered and dosing schedule can be readily determined by thoseof skill with routine experimentation by monitoring the blood plasmalevels of administered compound and/or an active metabolite thereof, andadjusting the dosage or dosing schedule as necessary. Generally, betweenabout 10 mg to about 3000 mg of active ingredient per dosage unit isused. This amount is more preferably between about 250 mg to about 1500mg of active ingredient, and most preferably between about 400 mg toabout 1000 mg of active ingredient per dosage unit.

[0053] 5.2 Hydrotropes

[0054] Examples of hydrotropes which can be used within the formulationsof the present invention include, but are not limited to, sorbitol,mannitol, nicotinic acid, nicotinamide, 2,5-dihydroxybenzoic acid,ascorbic acid, ascorbyl dipalmitate, fructose, glucose, glucoseglutamate, glucuronic acid, glycerin, 1,2,6-hexanetriol, hydroxystearylmethylglucamine, inositol, lactose, maltitol, sorbeth-20, sucrose,thioglycerin, tris(hydroxymethyl)nitromethane, tromethamine and xylitol.A preferred group of suitable hydrotropes are polyhydroxylated alcohols.The preferred polyhydroxylated alcohol is sorbitol.

[0055] While not intending to be bound by theory, it is believed that anincrease in chemical and biological properties in pharmaceuticalcompositions comprising an aminoalkyl phosphorothioate and a hydrotropewill be, in part, due to the enhanced solubility and rate of dissolutionof aminoalkyl phosphorothioate compounds in hydrotropes. Sorbitol, apolyhydroxylated alcohol, can be utilized in pharmaceutical preparationsto increase the absorption of vitamins and other nutrients. Sorbitol isapproved for use as a sweetener and as a osmotic diuretic. Sorbitol isalso used as a stabilizer for drug, vitamin and antacid suspensions. Thepresent invention is based, in part, on the quite unexpected discoverythat hydrotropes, including polyhydroxylated alcohols, and sorbitol inparticular, enhance the solubility and rate of diffusion of aminoalkylphosphorothioate compounds at relatively high concentrations.Hydrotropes may also be effective by enabling a selective transport ofthe aminoalkyl phosphorothioate compounds through tight junctions bymeans of passive diffusion.

[0056] The amount of hydrotrope used in the pharmaceutical compositionsof the invention depends on the specific hydrotrope or blend ofhydrotropes selected. However, the hydrotrope is preferably used at aconcentration of from about 0.5 mg/ml to about 100 mg/ml. Morepreferably, the hydrotrope is used at a concentration of from about 10mg/ml to about 30 mg/ml. Most preferably the hydrotrope is used at aconcentration of about 20 mg/ml. In a preferred embodiment, the amountof hydrotrope used is such as to lead to an effective concentration ofamifostine of between about 25 mg/ml and 300 mg/ml. Preferredhydrotropes include, but are not limited to nicotinic acid andnicotinamide, and polyhydroxylated alcohols such as sorbitol, mannitoland 2,5-dihydroxybenzoic acid (gentisic acid). A most preferredhydrotrope is sorbitol. In a preferred embodiment, sorbitol is used at aconcentration of about 20 mg/ml.

[0057] 5.3 Chelating Agents

[0058] Examples of chelating agents that can be used within theformulations of the present invention include, but are not limited to,aminotrimethylene phosphoric acid, calcium disodium ethylenediaminetetraacetic acid (EDTA), citric acid, cyclohexanediamine tetraaceticacid, diammonium citrate, diethylenetriamine pentacetic acid (DPTA),dipotassium EDTA, disodium EDTA, disodium pyrophosphate, EDTA, etidronicacid, glucuronic acid, pentapotassium triphosphate, pentasodiumaminotrimethylene phosphonate, pentasodium pentetate, pentasodiumtriphosphate, pentetic acid, sodium citrate, sodiumdihydroxyethylglycinate, sodium gluconate, sodium hexametaphosphate,sodium metaphosphate, sodium metasilicate, sodium trimetaphosphate,triethanolamine EDTA, tetrahydroxypropyl ethylenediamine, tetrapotassiumpyrophosphate, tetrasodium EDTA, tetrasodium etidronate, tetrasodiumpyrophosphate, tripotassium EDTA, trisodium EDTA, trisodium NTA andtrisodium phosphate. Preferred chelating agents are DPTA and sodiumsalts of EDTA. The most preferred chelating agent is EDTA.

[0059] Chelating agents, which are also known as sequestrants, arecompounds which can form stable complexes with metal ions, and thusprevent adverse effects on the stability or appearance of formulations.It was found that the chelating agents unexpectedly demonstrate theability to enhance the biological activity of the aminoalkylphosphorothioate compounds of the present invention.

[0060] The amount of chelating agent used in the pharmaceuticalcompositions of the invention depends on the specific chelating agent orblend of chelating agents selected. However, the chelating agent ispreferably used at a concentration of from about 0.5 mg/ml to about 100mg/ml. More preferably, the chelating agent is used at a concentrationof from about 10 mg/ml to about 30 mg/ml. Most preferably the chelatingagent is used at a concentration of 20 mg/ml. The amount of chelatingagent used is such as to lead to an effective concentration ofamifostine of between about 25 mg/ml and 300 mg/ml. Preferred chelatingagents include, but are not limited to ethylenediamine tetraacetic acid(EDTA) and diethylenetriamine pentaacetic acid (DTPA) A most preferredchelating agent is EDTA. In a preferred embodiment, EDTA is used at aconcentration of about 20 mg/ml.

[0061] 5.4 Surfactants

[0062] Examples of surfactants suitable for use in the formulations ofthe present invention include, but are not limited to, cholic acid andsalts of cholic acid, deoxycholic acid and salts of deoxycholic acid,taurocholic acid and salts of taurocholic acid, polyvinylpyrrolidone,PEG compounds such as cocamines, glyceryl stearates, glyceryl oleates,hydrogenated lanolins, lanolins, laurates and oleates, sorbitanlaurates, sorbitan palmitates, sorbitan stearates, quaterniumsurfactants, sodium sulfates, glyceryl compounds, palmitic acid and itsderivatives and oleic acid and its derivatives.

[0063] Additional suitable surfactants may be selected from theliterature by one skilled in the art. (See, e.g., SurfactantEncyclopedia, 2^(nd) Edition, M. M. Reiger (1996).)

[0064] A preferred subset of suitable surfactants are lipophilicsurfactants. Lipophilic surfactants are surface active compounds whichdemonstrate an affinity for lipid compounds, which are organic waxesoils and fats, of low solubility in water but soluble in organicsolvents. The surfactants of the present invention solubilize theaminoalkyl phosphorothioate compounds, thus forming a lipophilic moiety.Transcellular absorption of the aminoalkyl phosphorothioate compounds isbrought about by administration of the lipophilic moiety to the patient.The invention is based, in part, on the quite unexpected discovery thatpharmaceutical compositions comprising combinations of aminoalkylphosphorothioate compounds and surfactants exhibit improved biologicalproperties over compositions comprising only aminoalkyl phosphorothioatecompounds in a suitable vehicle.

[0065] The amount of surfactant used in the pharmaceutical compositionsof the invention depends on the specific surfactant or blend ofsurfactants selected. However, the surfactant is preferably used at aconcentration of from about 0.1 mg/ml to about 10 mg/ml. Morepreferably, the surfactant is used at a concentration of from about 0.3mg/ml to about 0.9 mg/ml. Most preferably the surfactant is used at aconcentration of from about 0.5 mg/ml to about 0.75 mg/ml. The amount ofsurfactant used is such as to lead to an effective concentration ofamifostine of between about 25 mg/ml and 300 mg/ml. Preferredsurfactants include, but are not limited to sodium deoxycholate, sodiumtaurocholate, TWEEN 80 and sodium dodecylsulfate. A most preferredsurfactant is sodium deoxycholate. In a preferred embodiment, TWEEN 80is used at a concentration of 1%. In another preferred embodiment,sodium dodecylsulfate is used at a concentration of 0.02%. In yetanother preferred embodiment, sodium deoxycholate is used at aconcentration of about 0.75 mg/ml.

[0066] Each of the above concentrations relating to the amount ofhydrotrope, chelating agent and surfactant used in the formulations ofthe invention is calculated for a single dose composition comprisingbetween about 10 mg to about 3000 mg of aminoalkyl phosphorothioate.This amount is more preferably between about 250 mg to about 1500 mg ofaminoalkyl phosphorothioate, and most preferably between about 400 mg toabout 1000 mg of aminoalkyl phosphorothioate per dosage unit.

[0067] 5.5 Uses of the Pharmaceutical Compositions

[0068] The pharmaceutical compositions of the present invention can beused to efficaciously treat patients suffering from any disorder that istreatable with aminoalkyl phosphorothioate compounds.

[0069] For example, the pharmaceutical compositions of the invention canbe used to selectively protect normal tissues against the toxicitiesassociated with ionizing radiation or chemotherapy in cancer patients.

[0070] The pharmaceutical compositions of the invention comprise activecompounds which are capable of stimulating bone marrow growth andcausing the bone marrow function to more rapidly recover followingchemotherapy. Thus, the pharmaceutical compositions of the inventionfurther provide a useful means for administering these active compoundsto patients suffering from diseases requiring bone marrow growth, suchas myelodysplastic syndrome (MDS), and to patients whose bone marrow hasbeen exposed to chemotherapy. The pharmaceutical compositions alsoprovide a useful means for administering the active compounds topatients suffering from human immunodeficiency virus (“HIV”) infection.

[0071] The preferred method of administration of the pharmaceuticalcompositions of the invention is by subcutaneous administration. Thisroute of administration provides numerous advantages over other typicalroutes of administration, such as intravenous or bolus injection. Onesignificant advantage is the reduction or decrease in undesirableside-effects suffered by patients receiving the therapy. Also, betterpatient compliance is achieved by use of subcutaneous administration, asintravenous administration is disliked by most patients. Further,subcutaneous administration does not necessarily require administrationby skilled practitioners, thus making therapy more convenient forpatients.

[0072] The benefits of subcutaneous administration coupled with thebenefits of the described formulations provide a unique and efficaciousprevention or treatment of a variety of diseases known to be treated byamifostine.

6. Administration and Preparation of Compositions

[0073] The pharmaceutical formulations described herein, orpharmaceutically acceptable salts and/or hydrates thereof, may beadministered singly, in combination with other compounds of theinvention, and/or in combination with other therapeutic agents,including cancer chemotherapeutic agents. The pharmaceuticalformulations may be administered alone or in an admixture with one ormore pharmaceutically acceptable carriers, excipients or diluents.Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

[0074] For injection, the formulations of the invention may be preparedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulations.

[0075] For oral administration, the formulations can be readily combinedwith pharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.Pharmaceutical preparations for oral use can be obtained solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

[0076] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycomprise gum arabic, talc, polyvinyl-pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of formulation or active compounddoses.

[0077] Pharmaceutical preparations which can be used orally include, butare not limited to push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can comprise the active ingredients inadmixture with filler such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

[0078] For buccal administration, the formulations may take the form oftablets or lozenges formulated in conventional manner.

[0079] For administration by inhalation, the formulations for useaccording to the present invention are conveniently delivered in theform of an aerosol spray from pressurized packs or a nebulizer, with theuse of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated to comprise a powder mix of theformulation and a suitable powder base such as lactose or starch.

[0080] The formulations may be formulated for parenteral administrationby injection, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Theformulations may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may comprise formulatory agents such assuspending, stabilizing and/or dispersing agents.

[0081] Pharmaceutical formulations for parenteral administrationinclude, but are not limited to aqueous solutions of the activecompounds in sterile water-soluble form. Additionally, suspensions ofthe pharmaceutical formulations may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles include,but are not limited to fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may comprise substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also comprisesuitable stabilizers or agents which increase the solubility of theformulations to allow for the preparation of highly concentratedsolutions.

[0082] Alternatively, the pharmaceutical formulations may be in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

[0083] The pharmaceutical formulations may also be used in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

[0084] In addition, the formulations may also be used as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations maycomprise suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

[0085] The pharmaceutical formulations also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

[0086] The formulations may be provided in a single, lyophilized dosageform, suitable for reconstitution and administration to a patient.Alternatively, the formulations may be provided in a form wherein thelyophilized aminoalkyl phosphorothioate active compounds are providedseparately from the remainder of the formulation, which remainder isadded to the active compound in the reconstitution step. The remainderof the formulation may be provided as a sterile aqueous solution, or inlyophilized form. The single dosage form may be prepared by loading theformulation into a vial and the vial into a freeze-drier which is thanevacuated. The vial then undergoes a freeze drying, or lyophilizationprocess which comprises freezing the composition, evacuating thelyophilization chamber and drying the frozen composition. The processmay further comprise further drying using a secondary drying stage,and/or a dessicating means such as the use of dessicants, a dessicator,desiccating stoppers, and the like. Upon completion of thelyophilization, an inert gas such as nitrogen is preferably added to thevials containing the lyophilized dosage form. The dosage form whereinthe aminoalkyl phosphorothioate active compounds are provided separatelyfrom the remainder of the formulation are prepared in a similar manner,with the active compounds being introduced into a vial forlyophilization, and the remainder of the formulation being introducedinto a second vial for lyophilization and/or liquid fill. Theformulations may further be provided in the form of a two-compartmentsyringe, or as a frozen solution of all the components of thecomposition.

[0087] 6.1 Effective Dosages of Active Materials

[0088] The pharmaceutical compositions of the present invention containthe active compounds of the composition in a therapeutically effectiveamount, i.e., an amount effective to achieve its intended prophylacticor therapeutic purpose. Of course, the actual amount of activeingredient will depend on, among other things, its intended purpose. Forexample, when administered to cancer patients as a cytoprotectant inconjunction with radiation or chemotherapy, such compositions willcomprise an amount of active ingredient effective to, inter alia,ameliorate the harmful effects of ionizing radiation or chemotherapeuticagents to normal tissues. When administered to patients suffering fromdiseases requiring bone marrow growth, such as MDS, or more rapidrecovery of bone marrow function following chemotherapy, suchcompositions will comprise an amount of active ingredient effective tostimulate bone marrow production or function, prevent the development ofor alleviate the existing symptoms of, or prolong the survival of, thepatient being treated. Determination of an effective amount is wellwithin the capabilities of those skilled in the art, especially in lightof the detailed disclosure herein.

[0089] For any active compound described herein the therapeuticallyeffective amount can be initially estimated from cell culture assays.For example, a dose can be formulated in animal models to achieve acirculating concentration range of compound, and/or an active metabolitethereof, that includes an effective concentration as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. See, e.g., Washburn et al., 1976, “Prediction ofthe Effective Radioprotective Dose of WR-2721 in Humans Through anInterspecies Tissue Distribution Study” Radiat. Res. 66:100-5. Further,dosages can be extrapolated from that known for amifostine (SeePhysicians' Desk Reference, 1999).

[0090] Therapeutically effective amounts for use in humans can also beestimated from animal models. For example, a dose for humans can beformulated to achieve a circulating concentration found to be effectivein animals.

[0091] A therapeutically effective dose can also be estimated from humanpharmacokinetic data. While not intending to be bound by any particulartheory, it is believed that efficacy is related to a subject's totalexposure to an applied dose of administered drug, and/or an activemetabolite thereof, as determined by measuring the area under the bloodconcentration-time curve (AUC). Thus, a dose that has an AUC ofadministered compound (and/or an active metabolite thereof) within about50% of the AUC of a dose known to be effective for the indication beingtreated is expected to be effective. A dose that has an AUC ofadministered compound (and/or an active metabolite thereof) within about70%, 80% or even 90% or more of the AUC of a known effective dose ispreferred. Adjusting the dose to achieve maximal efficacy in humansbased on the methods described above, particularly on the bloodconcentration and duration of administered compound and/or its activemetabolites is well within the capabilities of the ordinarily skilledartisan.

[0092] For use as a cytoprotectant to selectively protect against thetoxicities of ionizing radiation or chemotherapeutic agents, acirculating concentration of active compound of about 2 μM to 100 μM isexpected to be effective, with about 5 μM to 50 μM being preferred.Alternatively, or in addition, a tissue concentration of active compoundof about 4 μM to 2200 μM is expected to be effective, with about 20 μMto 350 μM being preferred.

[0093] For use as a radioprotectant against the toxicities of ionizingradiation or as a chemoprotectant against the toxicities of cancertherapy, the dose should be administered enough in advance of exposureto radiation or chemotherapy to provide effect. For i.v. administration,the dose is preferably administered within 30 min. prior to theadministration of radiation or chemotherapy. For subcutaneousadministration, the dose is preferably administered about 20 to 90minutes prior to the administration of radiation therapy.

[0094] For use in treating diseases requiring bone marrow growth, suchas MDS, or recovery of bone marrow function, a circulating concentrationof active compound of about 2 μM to 100 μM is expected to be effective.Alternatively, or in addition, a tissue concentration of active compoundof about 0.1 μM to 1000 μM is expected to be effective, with about 10 μMto 500 μM being preferred.

[0095] For subcutaneous administration of amifostine patient dosagesusually range from about 50 mg/day to 1500 mg/day, commonly from about100 mg/day to 1100 mg/day and typically from about 200 mg/day to 1000mg/day. Stated in terms of body weight, usual dosages range from 0.5mg/kg/day to 20 mg/kg/day, commonly from about 1.1 mg/kg/day to 18mg/kg/day and typically from about 2.2 mg/kg/day to 16.2 mg/kg/day.Stated in terms of patient body surface areas, usual doses range fromabout 22 mg/m²/day to 800 mg/m²/day, commonly from about 45 mg/m²/day to720 mg/m²/day and typically from about 90 mg/m²/day to 650 mg/m²/day.

7. EXAMPLE 1 Subcutaneous Administration of Amifostine Protected AnimalsAgainst Radiation-Induced Mucositis

[0096] One of the major limiting acute toxicities associated withradiotherapy is radiation-induced mucositis. The ability to reduce theduration and severity of acute mucosal reactions is of particularimportance in the radiotherapy and/or chemotherapy of head and neckcancer. Therefore, the radioprotective effects of amifostine wereexamined in an experimental model of mucositis. In particular, the studycompared the radioprotective effects of amifostine by subcutaneous(s.c.) and intraperitoneal (i.p.) administration. The mouse modeldeveloped by Parkins et al. was used to examine the mucosal reactions inthe inferior lip of mice after irradiation, and this model has beenestablished as a reproducible model in the art (Parkins et al., 1983,Radiother. Oncol. 1:159-165).

[0097] 7.1 Experimental Design

[0098] C57BL/6 female mice of 8-10 weeks old were used and fed with semiliquid food. The mice were randomly divided into treatment groups offive mice each. The treatment groups were, for example,

[0099] Group 1: Saline solution (s.c.) and irradiation

[0100] Group 2: Amifostine, saline solution, (s.c.) and irradiation

[0101] Group 3: Amifostine, 2% EDTA and 2% Sorbitol and irradiation(s.c.)

[0102] Group 4: Amifostine and 0.075% Sodium Deoxycholate andirradiation (s.c.)

[0103] Group 5: Saline solution (s.c.), no irradiation

[0104] Group 6: Amifostine and 0.2% EDTA and irradiation (s.c.)

[0105] (See also Tables Below)

[0106] Unanesthetized mice were maintained in supine position andirradiated exclusively on the tip of their mouth. They were immobilizedusing jigs comparable to those previously used by Ang et al. (1982, Int.J. Radiat. Oncol. Biol. Phys. 8:145-148). Irradiation was performed witha RT 250 Philips apparatus delivering 1.98 Gy per min. (200 Kv, 20 mA,filter of 0.2 mm de Cu). During irradiation, a constant normobaric airrenewal was maintained. The effects of amifostine were evaluated using asingle dose of 16.5 Gy.

[0107] 7.2 Administration of Amifostine

[0108] Amifostine was dissolved in appropriate diluent to achieve afinal concentration of 50 mg/ml immediately before injections.Amifostine was dissolved in 0.075% sodium deoxycholate to achieve afinal concentration of 50 mg/ml. Amifostine was dissolved in a mixtureof 2% EDTA and 2% sorbitol to achieve a final concentration of 50 mg/ml.Amifostine was dissolved in 0.2% EDTA to achieve a final concentrationof 50 mg/ml. Subcutaneous injections (200 mg/Kg) were conducted 30minutes before irradiation. Where a control group was used, a firstcontrol group was injected with the saline solution and subsequentlyirradiated, and a second control group was injected with the salinesolution, but was not irradiated.

[0109] 7.3 Mucositis Scoring System

[0110] The effects of irradiation on lip mucosa were evaluated using thescoring system described by Parkins et al. (1983, Radiother. Oncol.1:159-165). Body weight of the treated mice were scored periodicallyafter treatment. Reduction in body weight was used as an objectiveindication of the severity of mucositis induced by irradiation,presumably resulting from the inability of the animals to eat. In thismodel, the acute reactions peaked on day 10 to 11 after irradiation.

[0111] Other symptoms of mucositis such as mucosal erythema and edemawere also recorded. These symptoms developed more slowly than weightloss following irradiation. Mucosal erythema and edema were scoredseparately, and could be analyzed as separate scores or as a combinedscore yielding a maximum score of 7. Mouse lip mucosal erythema wasscored according to Table 1. TABLE 1 Scoring System for Mucosal ErythemaScore Mucosal Observation 0.5 doubtful if abnormally pink 1 slight butdefinitely reddening 2 severe reddening 3 focal desquamation 4 exudationor crusting covering about ½ lip area 5 exudation or crusting coveringmore than ½ lip area

[0112] Mucosal edema (swelling) of the lips was scored according totable 2. TABLE 2 Scoring System for Mucosal Edema Score MucosalObservation 0.5 50—50 doubtful if any swelling 1 slight but definitelyswelling 2 severe swelling

[0113] 7.4 Results

[0114] Body weight reduction of irradiated mice was measured as anobjective indicator of mucositis. A single dose of irradiation greatlyreduced the body weight of the animals (FIG. 1). Non-irradiated animalsmaintained steady body weight throughout the course of the study. Lesssevere reductions in body weight were observed in animals which receivedsubcutaneous injections of amifostine as compared to irradiation andsaline injection.

[0115] Non-irradiated mice had no mucositis during the entire period ofthe experiment. In contrast, mucositis was observed in all irradiationgroups.

[0116] The body weight and total mucositis score of mice followingadministration of a formulation comprising amifostine, compared to thebody weight and total mucositis score of mice in the two control groups(one group being administered saline solution and no irradiation, thesecond being administered saline solution followed by irradiation) areshown in Table 3. TABLE 3 Amifostine and Saline Solution and SalineSolution Radiotherapy Radiotherapy Day Weight Score Weight Score WeightScore 0 16.3 0.0 16.4 0.0 16.6 0.0 6 17.0 0.0 15.8 0.5 15.7 1.8 10 17.50.0 17.4 1.7 13.8 4.6 11 18.2 0.0 17.8 1.8 14.8 5.7 12 18.0 0.0 17.6 1.315.1 4.6 18 18.7 0.0 18.4 0.4 17.5 3.6 28 19.9 0.0 19.5 0.0 19.4 1.6

[0117] The data in Table 3 are presented in graphical form in FIGS. 1Aand 1B, wherein the total mucositis score and the weight change arepresented.

[0118] The body weight and total mucositis score of mice receiving theformulation comprising amifostine and 0.075% sodium deoxycholate areshown in Table 4. TABLE 4 Amifostine with 0.075% Sodium Deoxycholate DayWeight Score 0 17.6 0.0 6 17.4 0.2 10 18.2 0.5 11 18.9 0.8 12 19.1 0.417 19.1 0.1 25 20.2 0.0

[0119] The data in Table 4 are presented in graphical form in FIGS. 2Aand 2B, wherein the total mucositis score and the weight change arepresented.

[0120] The weight and total mucositis score of mice receiving theformulation comprising amifostine, 2% EDTA and 2% shown in Table 5.TABLE 5 Amifostine with 2% Edetate Disodium, 2% Sorbitol; pH = 7 DayWeight Score 0 17.8 0.0 6 17.2 0.2 10 17.3 1.2 11 18.2 1.4 12 18.3 1.117 19.1 0.8 25 19.8 0.6

[0121] The data in Table 5 are presented in graphical form in FIG. 3,wherein the total mucositis score and the body weight are presented.

[0122] The body weight of mice following administration of a formulationcomprising amifostine, compared to the body weight of mice followingadministration of a formulation comprising amifostine and 0.2% EDTA areshown in Table 6. TABLE 6 Amifostine Amifostine with pH = 7 0.2% EDTADay Weight Score Weight Score 0 16.5 0.0 16.9 0.0 6 16.0 0.7 16.7 0.5 1016.9 2.4 17.3 2.0 11 17.3 3.2 17.8 2.1 12 17.3 1.4 17.6 1.5 18 18.2 0.518.7 0.1 28 19.5 0.0 20.0 0.0

[0123] The data in Table 6 are presented in graphical form in FIG. 4,wherein the total mucositis score and body weight of the twoformulations are presented.

[0124] The body weight of mice following administration of a formulationcomprising amifostine, compared to the body weight of mice followingadministration of a formulation comprising amifostine, 2% EDTA and 2%sorbitol, and the body weight of mice following administration of aformulation comprising amifostine and 0.075% sodium deoxycholate areshown in Table 7. TABLE 7 Amifostine with Amifostine with Amifostine 2%Edetate Disodium 0.075% Sodium pH = 7 2% Sorbitol, pH = 7 DeoxycholateDay Weight Score Weight Score Weight Score 0 16.9 0.0 17.8 0.0 17.6 0.06 16.5 0.2 17.2 0.2 17.4 0.2 10 16.5 1.6 17.3 1.2 18.2 0.5 11 17.6 1.618.2 1.4 18.9 0.8 12 18.0 0.9 18.3 1.1 19.1 0.4 17 17.8 0.3 19.1 0.819.1 0.1 25 18.7 0.1 19.8 0.6 20.2 0.0

[0125] The data in Table 7 are presented in graphical form in FIG. 5,wherein the total mucositis score and the body weight of theformulations are presented.

[0126] The data presented in FIGS. 3, 4 and 5 show that subcutaneousadministration of amifostine formulations was effective in reducing theadverse effects of radiation, as measured by the change in body weightand the visual appearance of mucositis. Furthermore, the subcutaneousadministration of the formulation comprising amifostine and sodiumdeoxycholate produced the highest efficacy at the time point of maximaladverse effects, day 10 for body weight (FIG. 5). The formulationcomprising amifostine with EDTA and sorbitol also demonstrated goodprotective efficacy compared to a formulation comprising amifostinealone (FIG. 5).

[0127] A comparison of the relative effects of a formulation comprisingamifostine, a formulation comprising amifostine and 0.075% sodiumdeoxycholate, and a formulation comprising amifostine, 2% EDTA and 2%sorbitol is shown in FIG. 5. FIG. 5 clearly demonstrates the improvedefficacy of the combination formulations which combine amifostine over aformulation which comprises amifostine alone.

7.5 EXAMPLE 2

[0128] These examples describe pharmaceutical formulations of theinvention comprising amifostine in combination with surfactants and/orhydrotropes and/or chelating agents. The pharmaceutical formulationswere administered, and the effects of irradiation on lip mucosaevaluated using the methods described in the first Example above. TABLE8 Formulations With Total Mucositis % Weight Change Amifostine (WR-2721)Score After 11 days After 6 Days Control (no radiation) 0.0  +5.8 0.075%Sodium 0.8  −1.1 Deoxycholate 2% Sorbitol 0.9 (1.8)*  −1.7 2% EdetateDisodium 1.4  −3.6 2% Sorbitol, pH = 7 pH = 7 1.6  −2.4 2% PEG 400 1.7(2.0)*  −3.6 2% Edetate Disodium 1.9  −2.4 2% Sorbitol, pH = 8 2%Edetate Disodium, 2.1  0.0 (−0.6)* pH = 7 pH= 8 2.2  −5.4 2% EdetateDisodium, 2.5 (2.7)*  −3.0 (−3.6)* pH = 8 pH = 8.5 3.1  −4.2 (−8.3)*0.9% Saline Solution 5.8 −11.4 (−21.6)*

[0129] TABLE 9 Formulations With Total Mucositis % Weight ChangeAmifostine (WR-2721) Score After 11 days After 6 Days Control (noradiation) 0.0  +4.3 0.2% Edetate Disodium 2.1  −1.2 pH = 8 2.9  −2.4 pH= 7 3.2  −3.0 pH = 6 3.2  −5.4 1% Tween-80 2.4  −4.1 1% Choline Chloride2.7  −8.7 0.02% SDS 2.4 −11.9 WR-1065 5.1  −8.7 0.9% Saline Solution 4.6 −5.4 (−16.8)*

[0130] The most beneficial results are obtained when the mucositisevaluation score is closest to those obtained in the control group whichreceiving no radiation. These studies demonstrate that the best resultsare found in a formulation which includes amifostine and 0.075% sodiumdeoxycholate.

7.6 EXAMPLE 3 Dissolution Studies

[0131] The ease of dissolution of amifostine in different formulationswas studied. Two sets of studies were carried out, the first using 2.2ml of diluent to reconstitute the amifostine, the second using 2.0 ml ofdiluent. 500 mg of amifostine per vial was used in each study. TABLE 10Amifostine with 2.2 ml of (Solubility) Reconstitution diluent Time(Sec.) at 25° C. 2% Sorbitol, pH = 8 15-20 2% Sorbitol 15-30 2%Sorbitol, 2% EDTA 30-45 Disodium, pH = 7 0.075% Sodium Deoxycholate45-60 0.05% Sodium Deoxycholate approx. 60 Water 45-60 0.9% NaCl 45-60

[0132] TABLE 11 Amifostine with 2.0 ml of (Solubility) Reconstitutiondiluent Time (Sec.) at 25° C. 2% Sorbitol, pH = 8 20-30 2% Sorbitol45-60 2% Sorbitol, 2% EDTA 60-75 Disodium, pH = 7 0.075% SodiumDeoxycholate 60-75 0.05% Sodium Deoxycholate  90-120 Water  90-120 0.9%NaCl  90-120

[0133] It can be seen that the ease of dissolution of amifostine in both2% sorbitol and a mixture of 2% sorbitol and 2% EDTA is greater thanthat in water.

[0134] The present invention is not to be limited in scope by theexemplified embodiments, which are intended as illustrations ofindividual aspects of the invention. Indeed, various modifications forthe invention in addition to those shown and described herein willbecome apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the-scope of the appended claims.

[0135] All publications cited herein are incorporated by reference intheir entireties for all purposes.

What is claimed is:
 1. A pharmaceutical composition comprising an amountof an aminoalkyl phosphorothioate and a surfactant which enhances thebiological activity of the composition.
 2. The pharmaceuticalcomposition of claim 1, wherein the amount of aminoalkylphosphorothioate is from about 10 mg to about 3000 mg.
 3. Thepharmaceutical composition of claim 1, wherein the amount of surfactantis added in the form of an aqueous dispersion, said dispersion having aconcentration of from about 0.1 mg/ml to about 20 mg/ml.
 4. Thepharmaceutical composition of claim 1, wherein the biological activityis cytoprotection, radio- or chemo-protection.
 5. The pharmaceuticalcomposition of claim 1, wherein the aminoalkyl phosphorothioate isamifostine or S-3-(3-methylaminopropylamino)-3-propyl dihydrogenphosphorothioate.
 6. The pharmaceutical composition of claim 4, whereinthe aminoalkyl phosphorothioate is amifostine.
 7. The pharmaceuticalcomposition of claim 1, wherein the surfactant is selected from thegroup consisting of cholic acid, a salt of cholic acid, deoxycholicacid, a salt of deoxycholic acid, taurocholic acid,polyvinylpyrrolidone, a salt of taurocholic acid, TWEEN 80 and sodiumdodecylsulfate.
 8. The pharmaceutical composition of claim 5, whereinthe surfactant is sodium deoxycholate.
 9. The pharmaceutical compositionof claim 1, wherein said composition is freeze-dried or lyophilized. 10.The pharmaceutical composition of claim 1, wherein said composition is aliquid.
 11. A pharmaceutical composition which comprises atherapeutically effective amount of amifostine and sodium deoxycholate.12. A pharmaceutical composition comprising an amount of an aminoalkylphosphorothioate and a hydrotrope.
 13. The pharmaceutical composition ofclaim 1, further comprising a hydrotrope.
 14. The pharmaceuticalcomposition of claim 13, wherein the hydrotrope is added in the form ofan aqueous solution, having a concentration of from about 0.5 mg/ml toabout 100 mg/ml.
 15. The pharmaceutical composition of claim 13, whereinthe hydrotrope is selected from the group consisting of sorbitol,mannitol, nicotinic acid, nicotinamide, 2,5-dihydroxybenzoic acid,ascorbic acid, ascorbyl dipalmitate, fructose, glucose, glucoseglutamate, glucuronic acid, glycerin, 1,2,6-hexanetriol, hydroxystearylmethylglucamine, inositol, lactose, maltitol, sorbeth-20, sucrose,thioglycerin, tris(hydroxymethyl)nitromethane, tromethamine and xylitol.16. The pharmaceutical composition of claim 13, wherein the hydrotropeis a polyhydroxylated alcohol.
 17. The pharmaceutical composition ofclaim 16, wherein the polyhydroxylated alcohol is sorbitol.
 18. Thepharmaceutical composition of claim 13, wherein said composition isfreeze-dried or lyophilized.
 19. The pharmaceutical composition of claim13, wherein said composition is a liquid.
 20. A pharmaceuticalcomposition comprising an amount of an aminoalkyl phosphorothioate, ahydrotrope and a chelating agent.
 21. The pharmaceutical composition ofclaim 20, wherein the chelating agent is added in the form of an aqueousdispersion, said dispersion having a concentration of from about 0.5mg/ml to about 100 mg/ml.
 22. The pharmaceutical composition of claim20, wherein the aminoalkyl phosphorothioate is amifostine orS-3-(3-methylaminopropylamino)-3-propyl dihydrogen phosphorothioate..23. The pharmaceutical composition of claim 20, wherein the hydrotropeis selected from the group consisting of sorbitol, mannitol, nicotinicacid, nicotinamide, 2,5-dihydroxybenzoic acid, ascorbic acid, ascorbyldipalmitate, fructose, glucose, glucose glutamate, glucuronic acid,glycerin, 1,2,6-hexanetriol, hydroxystearyl methylglucamine, inositol,lactose, maltitol, sorbeth-20, sucrose, thioglycerin,tris(hydroxymethyl)nitromethane, tromethamine and xylitol.
 24. Thepharmaceutical composition of claim 20, wherein the hydrotrope issorbitol.
 25. The pharmaceutical composition of claim 20, wherein thechelating agent is EDTA or DTPA.
 26. The pharmaceutical composition ofclaim 20, wherein the chelating agent is EDTA.
 27. The pharmaceuticalcomposition of claim 20, wherein said composition is freeze-dried orlyophilized.
 28. The pharmaceutical composition of claim 20, whereinsaid composition is a liquid.
 29. A pharmaceutical composition whichcomprises a therapeutically effective amount of amifostine, an amount ofsorbitol and an amount of EDTA.
 30. The pharmaceutical composition ofclaims 1 or 13, further comprising a chelating agent.
 31. Thepharmaceutical composition of claim 30, wherein the chelating agent isEDTA.
 32. The pharmaceutical composition of claim 30, wherein saidcomposition is freeze-dried or lyophilized.
 33. The pharmaceuticalcomposition of claim 30, wherein said composition is a liquid.
 34. Apharmaceutical composition comprising an amount of an aminoalkylphosphorothioate and a chelating agent, wherein said pharmaceuticalcomposition is adapted for use via subcutaneous administration.
 35. Thepharmaceutical composition of claim 34, wherein the aminoalkylphosphorothioate is amifostine orS-3-(3-methylaminopropylamino)-3-propyl dihydrogen phosphorothioate. 36.The pharmaceutical composition of claim 20, wherein the chelating agentis EDTA.
 37. A pharmaceutical composition which comprises atherapeutically effective amount of amifostine, an amount of sodiumdeoxycholate, an amount of sorbitol and an amount of EDTA.
 38. Thepharmaceutical composition of claim 1, 11, 12, 13, 20 or 34 wherein thecomposition has a pH of between about 6 and about
 8. 39. Thepharmaceutical composition of claim 1, wherein said pharmaceuticalcomposition is adapted for use via subcutaneous administration.
 40. Apharmaceutical composition adapted for use via subcutaneousadministration, comprising amifostine and a compound selected from thegroup consisting of 0.075% Sodium Deoxycholate, 2% Sorbitol, 2% EdetateDisodium, 0.2% Edetate Disodium, 1% Tween 80, 1% Choline Chloride, 0.02%Sodium Dodecyl Sulfate, WR-1065 and mixtures thereof, and wherein thecomposition has a pH of between about 6 and about
 8. 41. Thepharmaceutical composition of claim 1, wherein said pharmaceuticalcomposition is adapted for use via intravenous administration.
 42. Thepharmaceutical composition of claim 1, wherein said pharmaceuticalcomposition is adapted for use via oral administration.
 43. A unitdosage form which comprises a pharmaceutical composition of claim 1, 11,12, 13, 20, or
 34. 44. A method of treating a patient having cancer fromthe toxicities associated with radio- or chemotherapy, using thepharmaceutical composition of claim 1, 11, 12, 13, 20 or
 34. 45. Amethod of protecting a patient having cancer from the toxicitiesassociated with radio- or chemotherapy, using the pharmaceuticalcomposition of claim 1, 11, 12, 13, 20 or
 34. 46. A method of treating apatient having MDS, using the pharmaceutical composition of claim 1, 11,12, 13, 20 or
 34. 47. A method of protecting a patient having MDS, usingthe pharmaceutical composition of claim 1, 11, 12, 13, 20 or 34.